Anti-inflammatory agents

ABSTRACT

The invention provides compounds, compositions and uses of compounds of general formula (I) or (I′), or pharmaceutically acceptable salts thereof, which are 3-aminocaprolactam derivatives, for the preparation of a medicament intended to treat an inflammatory disorder.

This application is a U.S. National Stage Filing under 35 U.S.C. 371from International Application Number PCT/GB2005/003133, filed Aug. 10,2005 and published in English as WO 2006/016152 A1 on Feb. 16, 2006,which claims the benefit under 35 U.S.C. §119 of United Kingdomapplication serial number 0417863.8, filed Aug. 11, 2004, whichapplications and publication are incorporated herein by reference.

The invention relates to the use of 3-aminocaprolactam derivatives forpreparing a medicament intended to prevent or treat inflammatorydisorders.

Inflammation is an important component of physiological host defence.Increasingly, however, it is clear that temporally or spatiallyinappropriate inflammatory responses play a part in a wide range ofdiseases, including those with an obvious leukocyte component (such asautoimmune diseases, asthma or atherosclerosis) but also in diseasesthat have not traditionally been considered to involve leukocytes (suchas osteoporosis or Alzheimer's disease).

The chemokines are a large family of signalling molecules with homologyto interleukin-8 which have been implicated in regulating leukocytetrafficking both in physiological and pathological conditions. With morethan fifty ligands and twenty receptors involved in chemokinesignalling, the system has the requisite information density to addressleukocytes through the complex immune regulatory processes from the bonemarrow, to the periphery, then back through secondary lymphoid organs.However, this complexity of the chemokine system has at first hinderedpharmacological approaches to modulating inflammatory responses throughchemokine receptor blockade. It has proved difficult to determine whichchemokine receptor(s) should be inhibited to produce therapeutic benefitin a given inflammatory disease.

More recently, a family of agents which block signalling by a wide rangeof chemokines simultaneously has been described: Reckless et al.,Biochem J. (1999) 340:803-811. The first such agent, a peptide termed“Peptide 3”, was found to inhibit leukocyte migration induced by 5different chemokines, while leaving migration in response to otherchemoattractants (such as FMLP or TGF-beta) unaltered. This peptide, andits analogs such as NR58-3.14.3 (i.e. Sequence ID No. 1c(DCys-DGln-DIle-DTrp-DLys-DGlnDLys-DPro-DAsp-DLeu-DCys)-NH₂), arecollectively termed “Broad Spectrum Chemokine Inhibitors” (BSCIs).Grainger et al., Biochem. Pharm. 65 (2003) 1027-1034 have subsequentlyshown BSCIs to have potentially useful anti-inflammatory activity in arange of animal models of diseases. Interestingly, simultaneous blockadeof multiple chemokines is not apparently associated with acute orchronic toxicity, suggesting this approach may be a useful strategy fordeveloping new anti-inflammatory medications with similar benefits tosteroids but with reduced side-effects.

However, peptides and peptoid derivatives such as NR58-3.14.3, may notbe optimal for use in vivo. They are quite expensive to synthesise andhave relatively unfavourable pharmacokinetic and pharmacodynamicproperties. For example, NR58-3.14.3 is not orally bioavailable and iscleared from blood plasma with a half-life period of less than 30minutes after intravenous injection.

Two parallel strategies have been adopted to identify novel preparationswhich retain the anti-inflammatory properties of peptide 3 andNR58-3.14.3, but have improved characteristics for use aspharmaceuticals. Firstly, a series of peptide analogs have beendeveloped, some of which have longer plasma half-lives than NR58-3.14.3and which are considerably cheaper to synthesise. Secondly, a detailedstructure: activity analysis of the peptides has been carried out toidentify the key pharmacophores and design small non-peptidic structureswhich retain the beneficial properties of the original peptide.

This second approach yielded several structurally distinct series ofcompounds which retained the anti-inflammatory properties of thepeptides, including 16-amino and 16-aminoalkyl derivatives of thealkaloid yohimbine, as well as a range of N-substituted3-aminoglutarimides. (Reference: Fox et al., J Med Chem 45 (2002)360-370: WO 99/12968 and WO 00/42071.) All of these compounds arebroad-spectrum chemokine inhibitors which retain selectivity overnon-chemokine chemoattractants, and a number of them have been shown toblock acute inflammation in vivo.

The most potent and selective of these compounds was(S)-3-(undec-10-enoyl)-aminoglutarimide (NR58,4), which inhibitedchemokine-induced migration in vitro with an ED₅₀ of 5 nM. However,further studies revealed that the aminoglutarimide ring was susceptibleto enzymatic ring opening in serum. Consequently, for some applications(for example, where the inflammation under treatment is chronic, such asin autoimmune diseases) these compounds may not have optimal properties,and a more stable compound with similar anti-inflammatory properties maybe superior.

As an approach to identifying such stable analogs, various derivativesof (S)-3-(undec-10-enoyl)-aminoglutarimide have been tested for theirstability in serum. One such derivative, the 6-deoxo analog(S)-3-(undec-10-enoyl)-tetrahydropyridin-2-one, is completely stable inhuman serum for at least 7 days at 37° C., but has considerably reducedpotency compared with the parental molecule.

Amide derivatives of 3-aminocaprolactam have already been disclosed inthe art. For example:

-   -   Japanese patent application No. 09087331 describes        3-aminocaprolactam amide derivatives wherein the amide alkyl        side chain may contain from 2 to 30 carbon atoms. These        compounds have been presented as oil-gelating agents.    -   U.S. Pat. No. 6,395,282 describes immunogenic conjugates        comprising a carrier molecule coupled to an autoinducer of a        Gram negative bacteria, wherein said autoinducer can be a        3-aminocaprolactam amide derivative wherein the amide alkyl side        chain may contain up to 34 carbon atoms. However, a therapeutic        use is disclosed only for the conjugates and not for the        isolated amide derivative.    -   An article by Weiss et al. (Research Communications in        Psychology, Psychiatry and Behavior (1992), 17(3-4), 153-159)        discloses a series of 3-aminocaprolactam amide derivatives, and        among others 3-hexanamido-DL-ε-caprolactam and        3-dodecanamido-DL-ε-caprolactam. These compounds are presented        as having only an in vitro activity but no significant in vivo        effect.

In other words, though some alkyl amide derivatives of3-aminocaprolactam have certainly been known in the art, no actualpharmaceutical use has been described for 3-aminocaprolactam amidederivatives.

The invention provides the use of a compound of general formula (I), ora pharmaceutically acceptable salt thereof, for the preparation of amedicament intended to treat inflammatory disorder:

whereinX is —CO—Y—(R¹)_(n) or SO₂—Y—(R¹)_(n);Y is a cycloalkyl or polycyloalkyl group (such as an adamantyl,adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl group);or is a cycloalkenyl or polycycloalkenyl group;each R¹ is independently selected from hydrogen or an alkyl, haloalkyl,alkoxy, haloalkoxy, alkenyl, alkynyl or alkylamino radical of 1 to 20carbon atoms (for example of 5 to 20 carbon atoms, of 8 to 20 carbonatoms, of 9 to 20 carbon atoms, of 10 to 18 carbon atoms, of 12 to 18carbon atoms, of 13 to 18 carbon atoms, of 14 to 18 carbon atoms, of 13to 17 carbon atoms);or each R¹ is independently selected from fluoro, chloro, bromo, iodo,hydroxy, oxyalkyl, amino, aminoalkyl or aminodialkyl radical; andn is any integer from 1 to m, where m is the maximum number ofsubstitutions permissible on the cyclo-group Y.

Alternatively R¹ may be selected from a peptido radical, for examplehaving from 1 to 4 peptidic moieties linked together by peptide bonds(for example a peptido radical of 1 to 4 amino acid residues).

The carbon atom at position 3 of the caprolactam ring is asymmetric andconsequently, the compounds according to the present invention have twopossible enantiomeric forms, that is, the “R” and “S” configurations.The present invention encompasses the two enantiomeric forms and allcombinations of these forms, including the racemic “RS” mixtures. With aview to simplicity, when no specific configuration is shown in thestructural formulae, it should be understood that the two enantiomericforms and their mixtures are represented.

Preferably, the compounds of general formula (I) or pharmaceuticallyacceptable salts thereof used according to this aspect of the inventionwill be compounds of general formula (I′)

wherein X has the same meaning as above.

Preferably, the compounds of general formula (I) or (I′), or theirpharmaceutically acceptable salts, will be such that the ring or ringsof Y constrain the bond angles at the alpha-carbon to be essentiallytetrahedral (i.e. sp3 hybrid bonds). The “alpha carbon” is either at the2-position (relative to the amide carbonyl) or at the 1-position(relative to the sulfonamide sulfonyl group).

Any substituent R¹ may be a substituent at any permissible position onthe ring or rings of the cyclo-group Y. In particular it is to be notedthat the invention includes compounds in which the “alpha carbon” isboth part of the cyclo group and is itself substituted. The definitionof (R¹)_(n) encompasses compounds of the invention with no substitution(i.e. R¹=hydrogen), compounds of the invention with mono substitution(i.e. R¹ is not hydrogen and n=1), and also multiple substitution (i.e.at least two R¹ groups are not hydrogen and n=2 or more).

The invention also provides pharmaceutical compositions comprising, asactive ingredient, a compound of general formula (I), or apharmaceutically acceptable salt thereof, and at least onepharmaceutically acceptable excipient and/or carrier:

whereinX is —CO—Y—(R1)_(n) or SO₂—Y—(R¹)_(n);Y is a cycloalkyl or polycyloalkyl group (such as an adamantyl,adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl group);or is a cycloalkenyl or polycycloalkenyl group;each R¹ is independently selected from hydrogen or an alkyl, haloalkyl,alkoxy, haloalkoxy, alkenyl, alkynyl or alkylamino radical of 1 to 20carbon atoms (for example of 5 to 20 carbon atoms, of 8 to 20 carbonatoms, of 9 to 20 carbon atoms, of 10 to 18 carbon atoms, of 12 to 18carbon atoms, of 13 to 18 carbon atoms, of 14 to 18 carbon atoms, of 13to 17 carbon atoms);or each R¹ is independently selected from fluoro, chloro, bromo, iodo,hydroxy, oxyalkyl, amino, aminoalkyl or aminodialkyl radical; andn is any integer from 1 to m, where m is the maximum number ofsubstitutions permissible on the cyclo-group Y.

Alternatively R¹ may be selected from a peptido radical, for examplehaving from 1 to 4 peptidic moieties linked together by peptide bonds(for example a peptido radical of 1 to 4 amino acid residues).

Preferably, the compounds of general formula (I) or pharmaceuticallyacceptable salts thereof used according to this aspect of the inventionwill be compounds of general formula (I′)

wherein X has the same meaning as above.

By pharmaceutically acceptable salt is meant in particular the additionsalts of inorganic acids such as hydrochloride, hydrobromide,hydroiodide, sulphate, phosphate, diphosphate and nitrate or of organicacids such as acetate, maleate, fumarate, tartrate, succinate, citrate,lactate, methanesulphonate, p-toluenesulphonate, palmoate and stearate.Also within the scope of the present invention, when they can be used,are the salts formed from bases such as sodium or potassium hydroxide.For other examples of pharmaceutically acceptable salts, reference canbe made to “Salt selection for basic drugs”, Int. J. Pharm. (1986), 33,201-217.

The pharmaceutical composition can be in the form of a solid, forexample powders, granules, tablets, gelatin capsules, liposomes orsuppositories. Appropriate solid supports can be, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine and wax. Other appropriate pharmaceuticallyacceptable excipients and/or carriers will be known to those skilled inthe art.

The pharmaceutical compositions according to the invention can also bepresented in liquid form, for example, solutions, emulsions, suspensionsor syrups. Appropriate liquid supports can be, for example, water,organic solvents such as glycerol or glycols, as well as their mixtures,in varying proportions, in water.

The invention also provides compounds and salts thereof of generalformula (I)

whereinX is —CO—Y—(R¹)_(n) or SO₂—Y—(R¹)_(n);Y is a cycloalkyl or polycyloalkyl group (such as an adamantyl,adamantanemethyl, bicyclooctyl, cyclohexyl, cyclopropyl group);or is a cycloalkenyl or polycycloalkenyl group;each R¹ is independently selected from hydrogen or an alkyl, haloalkyl,alkoxy, haloalkoxy, alkenyl, alkynyl or alkylamino radical of 1 to 20carbon atoms (for example of 5 to 20 carbon atoms, of 8 to 20 carbonatoms, of 9 to 20 carbon atoms, of 10 to 18 carbon atoms, of 12 to 18carbon atoms, of 13 to 18 carbon atoms, of 14 to 18 carbon atoms, of 13to 17 carbon atoms);or each R¹ is independently selected from fluoro, chloro, bromo, iodo,hydroxy, oxyalkyl, amino, aminoalky or aminodialkyl radical; andn is any integer from 1 to m, where m is the maximum number ofsubstitutions permissible on the cyclo-group Y.

Alternatively R¹ may be selected from a peptido radical, for examplehaving from 1 to 4 peptidic moieties linked together by peptide bonds(for example a peptido radical of 1 to 4 amino acid residues).

Preferably, the compounds of general formula (I) or salts thereof usedaccording to this aspect of the invention will be compounds of generalformula (I′)

wherein X has the same meaning as above.

Preferably, the compounds of general formula (I) or (I′) when used inthe invention, or their salts, will be such that the ring or rings of Yconstrain the bond angles at the alpha-carbon to be essentiallytetrahedral (i.e. sp3 hybrid bonds).

In particular, preferred compounds of general formula (I) or (I′) andtheir salts according to any aspect of the present invention areselected from the group consisting of:

-   (S)-3-(Cyclohexanecarbonyl)amino-caprolactam;-   (S)-3-(1′-methylcyclohexanecarbonyl)amino-caprolactam;-   (S)-3-(Cyclohex-1′-enecarbonyl)amino-caprolactam;-   (S)-3-(trans-4′-pentylcyclohexane-1-carbonyl)amino-caprolactam;-   (S)-3-(4′-pentyl[2,2,2]bicyclo-octane-1-carbonyl)amino-caprolactam;-   (S)-3-(1′-Adamantanecarbonyl)amino-caprolactam;-   (S)-3-(1′-Adamantanylmethanecarbonyl)amino-caprolactam;-   (S)-3-(3′-chloro-1′-adamantanecarbonyl)amino-caprolactam;-   (S)-3-(3′,5′-Dimethyl-1′-adamantanecarbonyl)amino-caprolactam;-   (S)-3-(3′,5′,7′-Trimethyl-1′-adamantanecarbonyl)amino-caprolactam;    and the salts thereof.

The most preferred compound is(S)-3-(1′-Adamantanecarbonyl)amino-caprolactam and salts thereof.

The invention also provides the sulfonamide analogues of the exemplifiedcompounds: i.e. the sulfonyl-amino-caprolactam equivalents of the saidcompounds.

As mentioned in the discussion of prior art above, certain alkyl amidederivatives of 3-amino caprolactam may be known as compounds per se(though it is not presently known that any have been described as suchas pharmaceutical compositions or for medical use in ananti-inflammatory context).

The invention includes compounds, compositions and uses thereof asdefined, wherein the compound is in hydrated or solvated form.

The amide derivatives of 3-aminocaprolactam described here arefunctional BSCIs. They are relatively inexpensive to synthesise, usingfacile synthesis routes provided herein; they are stable in human serumand consequently have excellent pharmacokinetic properties; they areorally bioavailable; they are highly potent broad-spectrum chemokineinhibitors in vitro with excellent selectivity over non-chemokinechemoattractants; they are highly potent and effective anti-inflammatoryagents in vivo in rodent models of inflammation; their administration isnot associated with any significant acute toxicity at the dosesnecessary to achieve a maximal therapeutic effect. Taken together, theseproperties suggest that amide derivatives of 3-aminocaprolactamrepresent anti-inflammatory medications with advantages over previouslydescribed compounds.

In comparison to the prior art the improvement of the present inventionlies in the provision of the 3-aminocaprolactam moiety with a side chainhaving one or more alkyl/alkenyl rings to constrain the bond angles atthe alpha carbon of the side chain. Compounds of this invention aresignificantly superior to compounds with linear alkyl chains (whetheralkyl amides or alkyl sulfonamides).

Prior art peptides (such as NR58-3.14.3) have the disadvantages that:(a) they are expensive and require solid phase synthesis (at least forthe longer ones) and (b) they clear very quickly via the kidneys and (c)they are generally less potent.

The prior art aminoglutarimides are cheap, not cleared quickly via thekidneys and more potent BUT they do not show metabolic stability.

The improvement described here, the aminocaprolactams, are cheap, notcleared by the kidney and even more potent, and are also metabolicallystable.

According to this invention, inflammatory disorders intended to beprevented or treated by the compounds of general formula (I) or (I′) orthe pharmaceutically acceptable salts thereof or pharmaceuticalcompositions or medicaments containing them as active ingredientsinclude notably:

-   -   autoimmune diseases, for example such as multiple sclerosis;    -   vascular disorders including stroke, coronary artery diseases,        myocardial infarction, unstable angina pectoris, atherosclerosis        or vasculitis, e.g., Beheet's syndrome, giant cell arteritis,        polymyalgia rheumatica, Wegener's granulomatosis, ChurgStrauss        syndrome vasculitis, Henoch-Schönlein purpura and Kawasaki        disease;    -   viral infection or replication, e.g. infections due to or        replication of viruses including pox virus, herpes virus (e.g.,        Herpesvirus saimiri), cytomegalovirus (CMV) or lentivirus;    -   asthma;    -   osteoporosis; (low bone mineral density);    -   tumor growth;    -   rheumatoid arthritis;    -   organ transplant rejection and/or delayed graft or organ        function, e.g. in renal transplant patients;    -   a disorder characterised by an elevated TNF-α level;    -   psoriasis;    -   skin wounds;    -   disorders caused by intracellular parasites such as malaria or        tuberculosis;    -   allergies; or    -   Alzheimer's disease.

According to this invention, further inflammatory disorders include:

-   -   ALS;    -   fibrosis (particularly pulmonary fibrosis, but not limited to        fibrosis in the lung);    -   the formation of adhesions (particularly in the peritoneum and        pelvic region);    -   antigen induced recall response;    -   immune response suppression.

These clinical indications fall under the general definition ofinflammatory disorders or disorders characterized by elevated TNFβlevels.

Where legally permissible, the invention also provides a method oftreatment, amelioration or prophylaxis of the symptoms of aninflammatory disease (including an adverse inflammatory reaction to anyagent) by the administration to a patient of an anti-inflammatory amountof a compound, composition or medicament as claimed herein.

Administration of a medicament according to the invention can be carriedout by topical, oral, parenteral route, by intramuscular injection, etc.

The administration dose envisaged for a medicament according to theinvention is comprised between 0.1 mg and 10 g depending on the type ofactive compound used.

According to the invention, the compounds of general formula (I) or (I′)can be prepared using the processes described hereafter.

Preparation of the Compounds of General Formula (I) or (I′)

All the compounds of general formula (I′) or (I′) can be prepared easilyaccording to general methods known to the person skilled in the art.

Nevertheless, the following preferred synthetic route is proposed:

The reaction shown in Diagram 1 may be carried out, for example, inchloroform or dichloromethane. The most preferred reaction solvent isdichloromethane.

The above reaction is preferably carried out in the presence of a base,for example Na₂CO₃.

The above reaction may be carried out at ambient temperature (about 25°C.) or more generally at a temperature between 20 and 50° C.

DEFINITIONS

The term “about” refers to an interval around the considered value. Asused in this patent application, “about X” means an interval from Xminus 10% of X to X plus 10% of X, and preferably an interval from Xminus 5% of X to X plus 5% of X.

The use of a numerical range in this description is intendedunambiguously to include within the scope of the invention allindividual integers within the range and all the combinations of upperand lower limit numbers within the broadest scope of the given range.Hence, for example, the range of 1 to 20 carbon atoms specified inrespect of (inter alia) formula I is intended to include all integersbetween 4 and 20 and all sub-ranges of each combination of upper andlower numbers, whether exemplified explicitly or not.

As used herein, the term “comprising” is to be read as meaning bothcomprising and consisting of: Consequently, where the invention relatesto a “pharmaceutical composition comprising as active ingredient” acompound, this terminology is intended to cover both compositions inwhich other active ingredients may be present and also compositionswhich consist only of one active ingredient as defined.

The term “peptidic moieties” used herein is intended to include thefollowing 20 naturally-occurring proteogenic amino acid residues:

SYMBOL: MEANING Ala Alanine Cys Cysteine Asp Aspartic Acid Glu GlutamicAcid Phe Phenylalanine Gly Glycine His Histidine Ile Isoleucine LysLysine Leu Leucine Met Methionine Asn Asparagine Pro Proline GlnGlutamine Arg Arginine Ser Serine Thr Threonine Val Valine TrpTryptophan Tyr Tyrosine

Modified and unusual amino acid residues, as well as peptido-mimetics,are also intended to be encompassed within the definition of “peptidicmoieties”.

Unless otherwise defined, all the technical and scientific terms usedhere have the same meaning as that usually understood by an ordinaryspecialist in the field to which this invention belongs. Similarly, allthe publications, patent applications, all the patents and all otherreferences mentioned here are incorporated by way of reference (wherelegally permissible).

The following examples are presented in order to illustrate the aboveprocedures and should in no way be considered to limit the scope of theinvention.

FIGURES

FIG. 1 shows the chemical structure of examples of compounds accordingto the invention.

EXAMPLES General Procedure for the Synthesis of the Starting Compounds

The hydrochlorides of (R) and (S)-3-amino-caprolactam, and thehydro-pyrrolidine-5-carboxylates of (R,R) and (S,S)-3-amino-caprolactamwere synthesised according to literature (cf. Boyle et al., J. Org.Chem., (1979), 44, 4841-4847; Rezler et al., J. Med. Chem. (1997), 40,3508-3515).

Example 1 (S)-3-(Cyclohexanecarbonyl)amino-caprolactam

(S,S)-3-amino-caprolactam hydro-pyrrolidine-5-carboxylate 2 (5 mmol) andNa₂CO₃ (15 mmol) in water (25 ml) were added to a solution ofcyclohexanecarbonyl chloride (5 mmol) in dichloromethane (25 ml) atambient temperature and the reaction was stirred for 12 hours. Theorganic layer was then separated and the aqueous phase was extractedwith additional dichloromethane (2×25 ml). The combined organic layerswere dried over Na₂CO₃ and reduced in vacuo. The residue was purified byrecrystallisation from EtOAc/hexane to give the lactam (540 mg, 45%);m.p. (EtOAc/hexanes) 180-181° C.; [α]_(D) ²⁵ (c=1, CHCd₃)+42.0;ν_(max)/cm⁻¹ 3294 (NH), 1668, 1614 (CO), 1537 (NH); 8H (500 MHz, CDCl₃)6.89 (1H, d, J 5.5, CHNH), 6.51 (1H, br s, CH₂NH), 4.48 (1H, dd, J 11,6, CHNH), 3.30-3.17 (2H, m, CH₂NH), 2.11 (1H, tt, J 11.5, 3.5,(CH₂)CHCO), 2.01 (1H, br d, J 13, lactam ring CH), 1.98-1.92 (1H, m,lactam ring CH), 1.87-1.70 (6H, m, lactam ring CH ×2+cyhex CH ×4),1.66-1.59 (1H, m, cyhex CH), 1.47-1.30 (4H, br m, lactam ring CH×2+cyhex CH ×2) and 1.23-1.15 (3H, m, cyhex CH ×3); 6_(c) (125 MHz,CDCl₃) 175.9, 175.3 (CO), 51.8 (NHCHCO), 45.2 (CH), 42.1, 31.7, 29.6,29.4, 28.9, 27.9, 25.7 (×2), 25.6 (CH₂); m/z (M+C₁₃H₂₂N₂O₂ requires238.16813) 238.16768.

Example 2 (S)-3-(1′-methylcyclohexanecarbonyl)amino-caprolactam

(S,S)-3-amino-caprolactam hydro-pyrrolidine-5-carboxylate 2 (5 mmol) andNa₂CO₃ (15 mmol) in water (25 ml) were added to a solution of1-methylcyclohexanecarbonyl chloride (5 mmol) in dichloromethane (25 ml)at ambient temperature and the reaction was stirred for 12 hours. Theorganic layer was then separated and the aqueous phase was extractedwith additional dichloromethane (2×25 ml). The combined organic layerswere dried over Na₂CO₃ and reduced in vacuo. The residue was purified byrecrystallisation from EtOAc/hexane to give the lactam (540 mg, 43%);m.p. (EtOAc/hexanes) 168-169 ° C.; [α]_(D) ²⁵ (c=1, CHCl₃)+33.0;ν_(max)cm⁻¹ 3380, 3241 (NH), 1674, 1638 (CO), 1501 (NH); δ_(H) (500 MHz,CDCl₃) 7.12 (1H, d, J5, CHNH), 6.52 (1H, br s, CH₂NH), 4.48 (1H, ddd, J11, 5.5, 1.5 CHNH), 3.30-3.16 (2H, m, CH₂NH), 2.01 (1H, br d, J 13,lactam ring CH), 1.98-1.86 (3H, m, lactam ring CH+cyhex CH ×2),1.85-1.73 (2H, m, lactam ring CH ×2), 1.56-1.47 (2H, m, cyhex CH ×2),1.47-1.33 (5H, br m, lactam ring CH ×2+cyhex CH ×3) and 1.33-1.25 (3H,m, cyhex CH ×3); δc (125 MHz, CDCl₃) 176.9, 167.0 (CO), 52.0 (NHCHCO),42.5 (C quat), 42.1, 35.5 (×2), 31.6, 28.9, 27.9 (CH₂), 26.4 (CH₃),25.8, 22.9 (×2) (CH₂); m/Z (M³⁰C₁₄H₂₄N₂O₂ requires 252.18378) 252.18323.

Example 3 (S)-3-(Cyclohex-1′-enecarbonyl)amino-caprolactam

(S,S)-3-amino-caprolactam hydro-pyrrolidine-5-carboxylate 2 (5 mmol) andNa₂CO₃ (15 mmol) in water (25 ml) were added to a solution ofcyclohex-1-ene-1-carbonyl chloride (5 mmol) in dichloromethane (25 ml)at ambient temperature and the reaction was stirred for 12 hours. Theorganic layer was then separated and the aqueous phase was extractedwith additional dichloromethane (2×25 ml). The combined organic layerswere dried over Na₂CO₃ and reduced in vacuo. The residue was purified byrecrystallisation from EtOAc/hexanes to give the lactam (431 mg, 36%);m.p. (EtOAc/hexanes) 151-152° C.; [α]_(D) ²⁵ (c=1, CHCl₃)+57.5;ν_(max)/cm⁻¹ 3219 (NH), 1652, 1628 (C═O, C═C), 1515 (NH); 6H (500 MHz,CDCl₃) 7.12 (1H, d, J5, CHNH), 6.67 (1H, qn, J 1.5, CH═C), 6.52 (1H, brs, CH₂NH), 4.54 (1H, ddd, J 11, 5.5, 1.5 CHNH), 3.32-3.18 (2H, m,CH₂NH), 2.30-2.17 (2H, m, CH₂CH═C), 2.16-2.10 (2H, m, CH═CCH₂), 2.07(1H, br d, J 15, lactam ring CH), 2.00-1.92 (1H, m, lactam ring CH),1.87-1.76 (2H, m, lactam ring CH ×2), 1.68-1.60 (2H, m, cyhex CH ×2),1.60-1.52 (2H, m, cyhex CH ×2) and 1.50-1.31 (2H, br m, lactam ring CH×2); 6_(c) (125 MHz, CDCl₃) 175.9, 167.4 (CO), 134.0 (CH═C), 132.8(CH═C), 52.1 (NHCHCO), 42.1, 31.6, 28.9, 27.9, 25.3, 24.0, 22.1, 21.5(CH₂); m/z (M⁺C₁₃H₂₀N₂O₂ requires 236.15248) 236.15208.

Example 4 (S)-3-(trans-4′-pentylcyelohexane-1-carbonyl)amino-caprolactam

(S,S)-3-amino-caprolactam hydro-pyrrolidine-5-carboxylate 2 (7 mmol) andNa₂CO₃ (21 mmol) in water (25 ml) were added to a solution oftrans-4-pentylcyclohexane-1-carbonyl chloride (6 mmol) indichloromethane (25 ml) at ambient temperature and the reaction wasstirred for 12 hours. The organic layer was then separated and theaqueous phase was extracted with additional dichloromethane (2×25 ml).The combined organic layers were dried over Na₂CO₃ and reduced in vacuo.The residue was purified by recrystallisation from EtOAc/hexane to givethe lactam (977 mg, 53%); m.p. 182-184° C.; [α]_(D) ²⁵ (c=1, CHCl₃)+32.2; ν_(max)/cm⁻¹ 3326 (NH), 1670, 1636 (CO), 1511 (NH); δ_(H) (500MHz, CDCl₃) 6.91 (1H, d, J 5.5, CHNB), 6.87-6.70 (1H, br m, CH₂NH), 4.44(1H, ddd, J 11, 6.0, 1.5, CHNH), 3.28-3.15 (2H, m, CH₂NH), 2.08-1.90(3H, br m, ring CH ×2+(CH₂)₂CHCO), 1.88-1.72 (6H, m, ring CH+chainCH₂X₄), 1.45-1.28 (4H, br m, ring CH+chain CH₂×2+chain CH(CH₂)₃),1.27-1.07 (9H, br m, ring CH+chain CH₂X₈) and 0.90-0.79 (5H, m, chainCH₂+CH₃); δ_(C) (125 MHz, CDCl₃) 176.0, 175.3 (CO), 51.8 (NHCHCO), 45.4(CH), 41.0 (CH₂), 37.1 (CH₂), 36.9 (CH), 32.5, 32.4, 32.1, 31.7, 29.6,29.4, 28.9, 27.9, 26.5, 22.6 (CH₂) and 14.0 (CH₃); m/z (M⁺ C₁₉H₃₂N₂O₂requires 308.24638) 308.24566.

Example 5(S)-3-(4′-pentyl[2,2,2]bicyclo-octane-1-carbonyl)amino-caprolactam

(S,S)-3-amino-caprolactam hydro-pyrrolidine-5-carboxylate 2 (5.5 mmol)and Na₂CO₃ (16.5 mmol) in water (25 ml) were added to a solution oftrans-4-pentylcyclohexane-1-carbonyl chloride (4.4 mmol) indichloromethane (25 ml) at ambient temperature and the reaction wasstirred for 12 hours. The organic layer was then separated and theaqueous phase was extracted with additional dichloromethane (2×25 ml).The combined organic layers were dried over Na₂CO₃ and reduced in vacuo.The residue was purified by recrystallisation from EtOAc/hexane to givethe lactam (868 mg, 57%); m.p. 195-196° C.; [α]_(D) ²⁵ (c=1,CHCl₃)+28.7; ν_(max)/cm⁻¹ 3395, 3254 (NH), 1677, 1626 (CO), 1501 (NH);δ_(H) (500 MHz, CDCl₃) 6.98 (1H, d, J 5.5, CH₂NH), 6.77-6.63 (1H, br m,CH₂NH), 4.41 (1H, dd, J 11, 5.5, CBNH), 3.27-3.15 (2H, m, CH₂NH),2.00-1.88 (2H, br m, ring CH ×2), 1.81-1.73 (2H, br m, ring CH ×2), 1.69(6H, br t, J 7.5, chain CCH₂CH₂C ×6), 1.43-1.30 (8H, br m, ring CH×2+chain CCH₂CH₂C×6), 1.24 (2H, sext, J 7, CH₂CH₃), 1.19-1.07 (4H, m,CH₂CH₂CH₂CH₃) 1.05-0.98 (2H, m, CH₂Bu) and 0.82 (3H, t, J 7, CH₃); δ_(C)(125 MHz, CDCl₃) 177.4, 176.1 (CO), 51.9 (NHCHCO), 42.0, 41.2 (CH₂),39.0 (C quat), 32.7, 31.6, 30.6 (×3) (CH₂), 30.4 (C quat), 28.9, 28.8(×3), 27.9, 23.3, 22.6 (CH₂) and 14.0 (CH₃); m/z (M+C₂₀H₃₄N₂O₂ requires334.26203) 334.26352.

Example 6 (S)-3-(1′-Adamantanecarbonyl)amino-caprolactam

(S)-3-amino-caprolactam hydrochloride 2 (1 mmol) and Na₂CO₃ (3 mmol) inwater (15 ml) were added to a solution of 1-adamnantanecarbonyl chloride(1 mmol) in dichloromethane (15 ml) at ambient temperature and thereaction was stirred for 2 hours. The organic layer was then separatedand the aqueous phase was extracted with additional dichloromethane(2×25 ml). The combined organic layers were dried over Na₂CO₃ andreduced in vacuo. The residue was recrystallised from CH₂Cl₂/hexanes togive (5)-3-(1′-adarnantanecarbonyl)amino-caprolactam (171 mg, 59%); m.p.256-258° C.; [α]_(D) ²⁵ (c=1, CHCl₃)+29.5; ν_(max)/cm⁻¹ 3411, 3259 (NH),1678, 1626 (CO), 1505 (NH); δ_(H) (500 MHz, CDCl₃) 7.08 (1H, d, J 5.5,CHNH), 6.67 (1H, br s, CH₂NH), 4.47 (1H, ddd, J 11, 5.5, 1.5, CHNH),3.32-3.17 (2H, m, CH₂NH), 2.06-1.94 (5H, m, 2×ring CH+3×adamantane CH),1.90-1.75 (8H, m, 2×ring CH+3×adamantane CH₂), 1.72 (3H, br d, J 14.5,3×adamantane CHH), 1.68 (3H, br d, J 14.5, 3×adamantane CHH) and1.47-1.32 (2H, m, 2×ring CH); 5c (125 MHz, CDCl₃) 177.2, 175.9 (CO),51.9 (NHCHCO), 42.2 (CH₂N), 40.5 (CCO), 39.0 (3×CH₂ adamantane), 36.5(3×CH₂ adamantane), 31.7, 28.9, 28.0 (CH₂ lactam), 28.1 (3×CHadamantane); m/z (MH⁺ C₁₇H₂₇N₂O₂ requires 291.2073) 291.1994.

Example 7 (S)-3-(1′-Adamantanylmethanecarbonyl)amino-caprolactam

(S)-3-amino-caprolactam hydrochloride 2 (4 mmol) and Na₂CO₃ (12 mmol) inwater (50 ml) were added to a solution of 1-adamantanemethanecarbonylchloride (4 mmol) in dichloromethane (50 ml) at ambient temperature andthe reaction was stirred for 2 hours. The organic layer was thenseparated and the aqueous phase was extracted with additionaldichloromethane (2×50 ml). The combined organic layers were dried overNa₂SO₄ and reduced in vacuo. The residue was recrystallised fromEtOAc/hexane to give(S)-3-(1′-adamantanylmethanecarbonyl)amino-caprolactam, recrystallisedfrom EtOAc to give white crystals (688 mg, 56%); m.p. 258-260° C.;[α]_(D) ²⁵ (c=1, CHCl₃)+30.7; ν_(max)/cm⁻¹ 3409, 3255 (NH), 1682, 1611(CO), 1539 (NH); OH (500 MHz, CDCl₃) 6.82 (1H, d, J5.5, CHNH), 6.77 (1H,br t, J 5.5, CH₂NH), 4.48 (1H, ddd, J 11, 6, 1.5, CHNH), 3.28-3.14 (2H,m, CH₂NH), 2.04 (1H, br d, J 13.5, C-4H), 1.97-1.86 (6H, m,C-5H+3×adamantane CH+CH₂CO), 1.84-1.72 (2H, m, C-5H+ C-6H), 1.63 (3H, brd, J 12, adamantane 3×CH₂), 1.60-1.54 (9H, m, 9×adamantane CH₂) and1.47-1.27 (2H, m, C-4 H+ C-6H); 5c (125 MHz, CDCl₃) 175.9 (lactam CO),170.1 (amide CO), 52.0 (NHCHCO), 51.4 (CH₂CO), 42.6 (3×adamantane CH₂),42.0 (NCH₂), 36.7 (3×CH₂ adamantane), 32.7 (C_(quat) adamantane), 31.7(C-4), 28.8 (C-6), 28.6 (3×CH adamantane), 28.5 (C-5); m/z (M+C₁₈H₂₁N₂O₂requires 304.2151) 304.21430.

Example 8 (S)-3-(3′-chloro-1′-adamantanecarbonyl)amino-caprolactam

(S)-3-amino-caprolactam hydrochloride 2 (3 mmol) and Na₂CO₃ (9 mmol) inwater (15 ml) were added to a solution of 3-chloro-1-adamantanecarbonylchloride (3 mmol) in dichloromethane (15 ml) at ambient temperature andthe reaction was stirred for 12 hours. The organic layer was thenseparated and the aqueous phase was extracted with additionaldichloromethane (2×25 ml). The combined organic layers were dried overNa₂CO₃ and reduced in vacuo. The residue was recrystallised fromEtOAc/Hexane to give(S)-3-(3′-chloro-1′-adamantanecarbonyl)amino-caprolactam (621 mg, 64%);m.p. 204-206° C.; [α]_(D) ²⁵ (c=0.5, CHCd₃)+26.2; ν_(max)/cm⁻¹ 3411,3267 (NH), 1679, 1630 (CO), 1508 (NH); δ_(H) (500 MHz, CDCl₃) 7.07 (1H,d, J 5.5, CHNH), 6.65-6.44 (1H, br m, CH₂NH), 4.43 (1H, dd, J 11, 5.5,CHNH), 3.24-3.17 (2H, m, CH₂NH), 2.24 (2H, br s, adamantane CH), 2.20(2H, br s, adamantane CH), 2.12-2.03 (4H, m, adamantane CH), 2.02-1.91(2H, m, 2×lactam ring CH), 1.85-1.72 (6H, m, 2×ring CH+4×adamantane CH),1.66-1.55 (2H, m, 2×adamantane CH) and 1.45-1.31 (2H, m, 2×ring CH);δ_(C) (125 MHz, CDCl₃) 175.9, 174.9 (CO), 67.4 (CCl), 51.9 (NHCHCO),48.6, 46.2 (×2) (3×CH₂ adamantane), 44.5 (CCO), 42.1 (CH₂N), 37.4, 37.3,34.5 (3×CH₂ adamantane), 31.5 (CH₂ lactam), 31.1 (2×CH adamantane),28.8, 27.9 (CH₂ lactam); m/z (MH C₁₇H₂₆N₂O₂Cl requires 325.1683)325.1696.

Example 9 (S)-3-(3′,5′-Dimethyl-1′-adamantanecarbonyl)amino-caprolactam

(S)-3-amino-caprolactam hydrochloride 2 (1 mmol) and Na₂CO₃ (3 mmol) inwater (15 ml) were added to a solution of3,5-dimethyl-1-adamantanecarbonyl chloride (1 mmol) in dichloromethane(15 ml) at ambient temperature and the reaction was stirred for 12hours. The organic layer was then separated and the aqueous phase wasextracted with additional dichloromethane (2×25 ml). The combinedorganic layers were dried over Na₂CO₃ and reduced in vacuo. The residuewas recrystallised from hexane to give(S)-3-(3′,5′-dimethyl-1′-adamantanecarbonyl)amino-caprolactam (200 mg,63%); m.p. 157-158° C.; [α ]_(D)25 (c=0.5, CHCl₃)+26.8; ν_(max)/cm⁻¹³²⁰⁶ (NH), 1647 (CO), 1548 (NH); δ_(H) (500 MHz, CDCl₃) 7.05 (1H, d, J5.0, CHNH), 6.49-6.24 (1H, br m, CH₂NB), 4.45 (1H, ddd, J 11, 5.5, 1.5,CHNH), 3.30-3.16 (2H, m, CH₂NH), 2.12-2.07 (1H, m, adamantane CH),2.04-1.90 (2H, m, 2×lactam ring CH), 1.86-1.73 (2H, m, 2×lactam ringCH), 1.67 (2H, br s, 2×adamantane CH), 1.51-1.26 (10H, br m,8×adamantane CH+2×lactam ring CH) 1.17-1.09 (2H, m, adamantane CH) and0.81 (6H, s, 2×CH₃); 5c (125 MHz, CDCl₃) 176.9, 176.0 (CO), 51.9(NHCECO), 50.6, 45.2 (×2), 42.7 (×2) (CH₂ adamantane), 42.4 (CCO), 42.1(CH₂N), 37.7 (CH₂ adamantane), 31.6 (CH₂ lactam), 31.0 (2×CCH₃), 30.4,29.3 (CH₃), 28.9 and 27.9 (CH₂ lactam); m/z (MH⁺C₁₉H₃₁N₂O₂ requires319.2386) 319.2372.

Example 10(S)-3-(3′,5′,7′-Trimethyl-1′-adamantanecarbonyl)amino-caprolactam

(S)-3-amino-caprolactam hydrochloride 2 (1 mmol) and Na₂CO₃ (3 mmol) inwater (15 ml) were added to a solution of3,5,7-trimethyl-1-adamantanecarbonyl chloride (1 mmol) indichloromethane (15 ml) at ambient temperature and the reaction wasstirred for 12 hours. The organic layer was then separated and theaqueous phase was extracted with additional dichloromethane (2×25 ml).The combined organic layers were dried over Na₂CO₃ and reduced in vacuo.The residue was recrystallised from EtOAc/hexane to give(s)-3-(3′,5′,7′-trimethyl-1′-adamantanecarbonyl)amino-caprolactam (188mg, 56%); m.p. 177-178 C; [α]_(D) ²⁵ (c=0.5, CHCl₃)+25.6; ν_(max)/cm⁴¹3377, 3220 (NH), 1677, 1623 (CO), 1514 (NH); SH (500 MHz, CDCl₃) 7.06(1H, d, J5.0, CHNH), 6.40-6.15 (1H, br m, CH₂NH), 4.46 (1H, ddd, J 11,5.5, 1.5, CHNH), 3.32-3.17 (2H, m, CH₂NH), 2.03-1.92 (2H, m, 2×lactamring CH), 1.86-1.74 (2H, m, 2×lactam ring CH) 1.47-1.32 (5H, m, 2×ringCH+6×adamantane CH) 1.06 (3H, br d, J 12, 3×adamantane CJ1H), 1.04 (3H,br d, J12, 3×adamantane CHH) and 0.83 (9H, s, 3×CH₃); 5c (125 MHz,CDCl₃) 176.8, 176.0 (CO), 51.9 (NHCHCO), 50.0 (3×CH₂ adamantane), 44.6(3×CH₂ adamantane), 43.4 (CCO), 42.1 (CH₂N), 31.8 (3×CCH₃), 31.7 (CH₂lactam), 30.0 (3×CH₃), 28.9, 27.9 (CH₂ lactam); m/z (MH+C₂₀H₃₃N₂O₂requires 333.2542) 333.2528.

Pharmacological Study of the Products of the Invention

Inhibition of MCP-1 Induced Leukocyte Migration

Assay Principle

The biological activity of the compounds of the current invention may bedemonstrated using any of a broad range of functional assays ofleukocyte migration in vitro, including but not limited to Boydenchamber and related transwell migration assays, under-agarose migrationassays and direct visualisation chambers such as the Dunn Chamber. Forexample, to demonstrate the inhibition of leukocyte migration inresponse to chemokines (but not other chemoattractants) the 96-wellformat micro transwell assay system from Neuroprobe (Gaithersburg, Md.,USA) has been used. In principle, this assay consists of two chambersseparated by a porous membrane. The chemoattractant is placed in thelower compartment and the cells are placed in the upper compartment.After incubation for a period at 37° C. the cells move towards thechemoattractant, and the number of cells in the lower compartment isproportional to the chemoattractant activity (relative to a series ofcontrols).

This assay can be used with a range of different leukocyte populations.For example, freshly prepared human peripheral blood leukocytes may beused. Alternatively, leukocyte subsets may be prepared, includingpolymorphonuclear cells or lymphocytes or monocytes using methods wellknown to those skilled in the art such as density gradientcentrifugation or magnetic bead separations. Alternatively, immortalcell lines which have been extensively validated as models of humanperipheral blood leukocytes may be used, including, but not limited toTHP-1 cells as a model of monocytes or Jurkat cells as model of naive Tcells.

Although a range of conditions for the assay are acceptable todemonstrate the inhibition of chemokine-induced leukocyte migration, aspecific example is hereby provided.

Materials

The transwell migration systems are manufactured by Neuroprobe,Gaithersburg, Md., USA.

The plates used are ChemoTx plates (Neuroprobe 101-8) and 30 μl clearplates (Neuroprobe MP30).

Geys' Balanced Salt Solution is purchased from Sigma (Sigma G-9779).

Fatty acid-free BSA is purchased from Sigma (Sigma A-8806).

MTT, i.e. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,is purchased from Sigma (Sigma M-5655).

RPMI-1640 without phenol red is purchased from Sigma (Sigma R-8755).

The THP-1 cell line (European Cell culture Collection) were used as theleukocyte cell population.

Test Protocol

The following procedure is used for testing the invention compounds forMCP-1 induced leukocyte migration:

First, the cell suspension to be placed in the upper compartment isprepared. The THP-1 cells are pelleted by centrifugation (770×g; 4 mins)and washed with Geys Balanced Salt Solution with 1 mg/ml BSA (GBSS+BSA).This wash is then repeated, and the cells repelleted before beingresuspended in a small volume of GBSS+BSA for counting, for exampleusing a standard haemocytometer.

The volume of GBSS+BSA is then adjusted depending on the number of cellspresent so that the cells are at final density of 4.45×10⁶ cells per mlof GBSS+BSA. This ensures that there are 100,000 THP-1 cells in each 25μl of the solution that will be placed in the upper chamber of theplate.

To test a single compound for its ability to inhibit MCP-1 inducedmigration, it is necessary to prepare two lots of cells. The suspensionof THP-1 cells at 4.45×10⁶ cells/ml is divided into two pots. To one potthe inhibitor under test is added at an appropriate final concentration,in an appropriate vehicle (for example at 1 μM in not more than 1%DMSO). To the second pot an equal volume of GBSS+BSA plus vehicle asappropriate (e.g. not more than 1% DMSO) is added to act as a control.

Next, the chemoattractant solution to be placed in the lower compartmentis prepared. MCP-1 is diluted in GBSS+BSA to give a final concentrationof 25 ng/ml. This is divided into two pots, as for the cell suspension.To one pot, the test compound is added to the same final concentrationas was added to the cell suspension, while to the other pot an equalvolume of GBSS+BSA plus vehicle as appropriate (e.g. not more than 1%DMSO) is added.

Note that the volume of liquid that needs to be added to make theaddition of the text compound needs to be taken into account, whenestablishing the final concentration of MCP-1 in the solution for thelower compartment and the final concentration of cells in the uppercompartment.

Once the chemoattractant solutions for the lower wells and cellsolutions for the upper chambers have been prepared, the migrationchamber should be assembled. Place 29 μl of the appropriatechemoattractant solution into the lower well of the chamber. Assaysshould be performed with at least triplicate determinations of eachcondition. Once all the lower chambers have been filled, apply theporous membrane to the chamber in accordance with the manufacturer'sinstructions. Finally, apply 25 μl of the appropriate cell solution toeach upper chamber. A plastic lid is placed over the entire apparatus toprevent evaporation.

The assembled chamber is incubated at 37° C., 5% CO₂, for 2 hours. Asuspension of cells in GBSS+BSA is also incubated under identicalconditions in a tube. These cells will be used to construct a standardcurve for determining the number of cells that have migrated to thelower chamber under each condition.

At the end of the incubation, the liquid cell suspension is gentlyremoved from the upper chamber, and 20 μl of ice-cold 20 mM EDTA in PBSis added to the upper chamber, and the apparatus is incubated at 4° C.for 15 mins. This procedure causes any cells adhering to the undersideof the membrane to fall into the lower chamber.

After this incubation the filter is carefully flushed with GBSS+BSA towash off the EDTA, and then the filter is removed.

The number of cells migrated into the lower chamber under each conditioncan then be determined by a number of methods, including directcounting, labelling with fluorescent or radioactive markers or throughthe use of a vital dye. Typically, we utilise the vital dye MTT. 3 μl ofstock MTT solution are added to each well, and then the plate isincubated at 37° C. for 1-2 hours during which time dehydrogenaseenzymes within the cells convert the soluble MTT to an insoluble blueformazan product that can be quantified spectrophotometrically.

In parallel, an 8-point standard curve is set up. Starting with thenumber of cells added to each upper chamber (100,000) and going down in2-fold serial dilutions in GBSS+BSA, the cells are added to a plate in25 μl, with 3 μl of MTT stock solution added. The standard curve plateis incubated along side the migration plate.

At the end of this incubation, the liquid is carefully removed from thelower chambers, taking care not to disturb the precipitated formazanproduct. After allowing to air dry briefly, 20 μl of DMSO is added toeach lower chamber to solubilise the blue dye, and absorbance at 595 nmis determined using a 96-well plate reader. The absorbance of each wellis then interpolated to the standard curve to estimate the number ofcells in each lower chamber.

The MCP-1 stimulated migration is determined by subtracting the averagenumber of cells that reached the lower compartment in wells where noMCP-1 was added from the average number of cells that reached the lowercompartment where MCP-1 was present at 25 ng/ml.

The impact of the test substance is calculated by comparing theMCP-1-induced migration which occurred in the presence or absence ofvarious concentrations of the test substance. Typically, the inhibitionof migration is expressed as a percentage of the total MCP-1 inducedmigration which was blocked by the presence of the compound. For mostcompounds, a dose-response graph is constructed by determining theinhibition of MCP-1 induced migration which occurs at a range ofdifferent compound concentrations (typically ranging from 1 nM to 1 μMor higher in the case of poorly active compounds). The inhibitoryactivity of each compound is then expressed as the concentration ofcompound required to reduce the MCP-1-induced migration by 50% (the ED₅₀concentration).

Results

The compounds of examples 1 to 8 and 10 were tested and were shown tohave an ED₅₀ of 100 nM or less in this test.

Enantioselectivity

The (S)- and (R)-enantiomers of two different members of theaminocaprolactam series can be synthesised to determine whether thebiological activity showed enantioselectivity.

The dose-response curves for each of the compounds as inhibitors ofMCP-1 induced THP-1 cell migration can be determined using the transwellmigration assay.

For the application of the compounds of the present invention asanti-inflammatory agents in vivo it is preferable to use the pure(S)-enantiomer of the compound, rather than the racemic mixture of thetwo enantiomers or the pure (R)-enantiomer.

1. A pharmaceutical composition comprising, as active ingredient, acompound of formula (I) or a pharmaceutically acceptable salt thereof,and at least one of a pharmaceutically acceptable excipient and carrier:

wherein X is —CO—Y—(R¹)_(n) or SO₂—Y—(R¹)_(n); Y is a cycloalkyl,polycycloalkyl, or cycloalkenyl group, wherein Y includes no more than10 carbon atoms; each R¹ is independently selected from hydrogen, anC₁₋₂₀ alkyl, C₁₋₂₀ alkylamino of 1 to 20 carbon atoms, fluoro, chloro,bromo, iodo, or hydroxy radical; and n is any integer from 1 to m, wherem is the maximum number of substitutions permissible on the cyclo-groupY.
 2. The composition of claim 1 wherein the compound of formula (I) isa compound of formula (I′) or a pharmaceutically acceptable saltthereof:


3. A compound of formula (I):

wherein X is —CO—Y—(R¹)_(n) or SO₂—Y—(R¹)_(n); Y is a cycloalkyl,polycycloalkyl, or cycloalkenyl group, wherein Y includes no more than10 carbon atoms; each R¹ is independently selected from hydrogen, anC₁₋₂₀ alkyl, C₁₋₂₀ alkylamino of 1 to 20 carbon atoms, fluoro, chloro,bromo, iodo, or hydroxy radical; and n is any integer from 1 to m, wherem is the maximum number of substitutions permissible on the cyclo-groupY.
 4. The compound of claim 3 wherein the compound of formula (I) is acompound of formula (I′):


5. A compound of formula (I) or a pharmaceutically acceptable saltthereof:

wherein X is —CO—Y—(R¹)_(n) or SO₂—Y—(R¹)_(n); Y is a cycloalkyl,polycycloalkyl, or cycloalkenyl group, wherein Y includes no more than10 carbon atoms; each R¹ is independently hydrogen, an C₁₋₂₀ alkyl,C₁₋₂₀ alkylamino of 1 to 20 carbon atoms, fluoro, chloro, bromo, iodo,or hydroxy radical; and n is any integer from 1 to m, where m is themaximum number of substitutions permissible on the cyclo-group Y;wherein the ring or rings of Y constrain the bond angles at thestereogenic alpha-carbon of the caprolactam ring of formula (I) suchthat the stereogenic alpha-carbon is essentially tetrahedral.
 6. Acompound according to claim 3, wherein the compound is:(S)-3-(Cyclohexanecarbonyl)amino-caprolactam;(S)-3-(1′-methylcyclohexanecarbonyl)amino-caprolactam;(S)-3-(Cyclohex-1′-enecarbonyl)amino-caprolactam;(S)-3-(trans-4′-pentylcyclohexane-1-carbonyl)amino-caprolactam;(S)-3-(4′-pentyl[2,2,2]bicyclo-octane-1-carbonyl)amino-caprolactam;(S)-3-(1′-Adamantanecarbonyl)amino-caprolactam;(S)-3-(1′-Adamantanylmethanecarbonyl)amino-caprolactam;(S)-3-(3′-chloro-1′-adamantanecarbonyl)amino-caprolactam;(S)-3-(3′,5′-Dimethyl-1′-adamantanecarbonyl)amino-caprolactam;(S)-3-(3′,5′,7′-Trimethyl-1′-adamantanecarbonyl)amino-caprolactam; or asulfonyl analog thereof, or a pharmaceutically acceptable salt thereof.7. The compound of claim 6 wherein the compound is(S)-3-(1′-Adamantanecarbonyl)amino-caprolactam or a pharmaceuticallyacceptable salt thereof.
 8. The compound of claim 3 wherein thesubstituent R¹ is not a straight chain alkyl group.
 9. The compound ofclaim 3 wherein the substituent R¹ is hydrogen.
 10. The compound ofclaim 3 wherein the substituent R¹ is not an alkyl group.
 11. Apharmaceutically acceptable composition comprising, as an activeingredient, a compound of claim 5 or a pharmaceutically acceptable saltthereof, and at least one of a pharmaceutically acceptable excipient anda pharmaceutically acceptable carrier.